I. Technical Field
The present invention relates generally to the fields of microbiology and bacterial genetics. More particularly, it concerns the biosynthetic pathway for tautomycetin (TTN) and its use to create TTN analogs.
II. Related Art
Tautomycetin (TTN, 1) and tautomycin (TTM, 2) (FIG. 1) are potent cell-permeable inhibitors of protein phosphatases (PPs) PP-1 and PP-2A and are recognized as potent inducers of apoptosis. TTN, first isolated from Streptomyces griseochromogenes (Cheng et al., 1989; Cheng et al., 1987), displays nearly a 40-fold preference for PP-1 inhibition over PP-2A and is the most selective PP-1 inhibitor reported to date (Mitsuhashi et al., 2001). The PP-1 selectivity of TTN likely plays a role in the agent's extraordinary immunosuppressive activity (Shim et al., 2002) and sharply contrasts the PP-2A selective inhibition by the natural product okadaic acid, another potent phosphatase inhibitor, making it a particularly useful tool (Bialogan and takai, 1988). Indeed, TTN has been instrumental in dissecting the role of PP-1 in the MEK-ERK pathway (Lee et al., 2006). TTM, isolated from Streptomyces spiroverticillatus, shares significant structural features with TTN, and yet displays only a weak preference for PP-1 inhibition relative to PP-2A (Mitsuhashi et al., 2001).
The inventor previously cloned and sequenced the biosynthetic gene clusters for both TTN and TTM (Li et al., 2009; Li et al., 2008). In the case of the highly selective PP-1 inhibitor TTN, the ttn biosynthetic gene cluster from S. griseochromogenes was characterized and its involvement in TTN biosynthesis confirmed by gene inactivation and complementation experiments (Li et al., 2009). The ttn cluster was localized to a 79-kb DNA region, consisting of 19 open reading frames that encode two modular type I polyketide synthases (TtnAB), one type II thioesterase (TtnH), eight proteins for dialkylmaleic anhydride biosynthesis (TtnKLMNOPRS), four tailoring enzymes (Tt-nCDFI), two regulatory proteins (TtnGQ), and one resistance protein (TtnJ). On the basis of functional assignments for each gene in the ttn cluster obtained from sequence analysis, the inventor formulated a model for biosynthesis of TTN that agrees well with previous feeding experiments, has been supported by in vivo gene inactivation experiments, and is supported by analogy to the recently reported ttm cluster. These findings set the stage to fully interrogate biosynthesis of TTN.
Of particular interest is the means by which the C222-C5 component (right hemisphere) is installed (FIG. 1). This component of TTN differs significantly from the corresponding right hemisphere of TTM and has been proposed as a crucial determinant dictating the greater PP-1 selectivity of TTN relative to TTM (Oikawa, 2002; Nishiyama et al., 1996; Sheppeck et al., 1997; Takai et al., 2000). This postulate has been substantiated by the recent crystal structure elucidation of PP-1 bound to TTM, although high-resolution structural information relating to PP-1 inhibition by TTN remains elusive (Kelker et al., 2009). Both TTN and TTM exist as equilibrating mixtures of anhydride and ring-opened diacids (Cheng et al., 1987; Cheng et al., 1990a; Cheng et al., 1990b); the PP-1-to-TTM crystal structure reveals that the diacid form of TTM is the active PP-1 inhibitor and implies, by analogy, that the diacid form of anhydride TTN is the species directly responsible for PP-1 inhibition (Kelker et al., 2009). The further understanding of the structure-function relationship of these compounds, as well as further analogs thereof, would be of considerable interest both from an academic standpoint as well as in the development of compounds for using in treating diseases such as cancer and autoimmune disfunction.